Tools for functional assessment of human cryptosporidiosis are still inadequate. Advances in stem cell-based organoid technologies resembling the primary intestinal epithelium of humans supports full life cycle progression of the parasite and allows new applications toward the comparison of the pathogenicity of different species or genotypes.

The overall aim of the project is to compare the pathogenicity of major Cryptosporidium genotypes circulating in Germany in stem cell derived organoid systems. In a pilot study, we evaluated the most common Cryptosporidium types in humans in Germany. We furthermore established an experimental infection model based on a previously described human stem cell derived intestinal organoid system.

Cryptosporidium positive samples were characterized by PCR for species determination and genotyping. Experimental infection of human small intestinal organoid derived monolayer (ODM) were performed with C. parvum gp60 genotype IIaA15G2R1 and evaluated by qPCR, immunofluorescence analysis (IFA) and by electron microscopy.

Of 125 Cryptosporidium positive human cases, 28% were determined as C. hominis and 65% as C. parvum. Gp60 typing revealed a variety of genotypes for both C. hominis (dominant type Ib10G2) and C. parvum (dominant type IIaA15G2R1). Evaluation of experimental infection of ODMs with sporozoites by qPCR showed approximately 10-fold increase of genome equivalents after 6 days post infection. The IFA analysis confirmed parasite infection and proliferation within the brush border of enterocytes. In the EM analysis trophozoites and meront stages were detected after 24 hours whereas sexual stages could be observed after 72 hours.

Our pilot study provides a first overview of dominant C. hominis and C. parvum genotypes circulating in Germany. We confirmed the suitability of our well-established ODM model for Cryptosporidium infection. Next steps will include more systematic sampling of human cryptosporidiosis cases in Germany and we will further pursue the ODM infection model to unravel the molecular basis of host-parasite interaction.